Project 2 (Replication-Associated Repair and Replication Fork Maintenance) of the SBDR Program Project focuses on the integration of multiple DNA repair pathways at replication forks and their roles in the maintenance of genomic stability. Cells devote significant resources to detecting and repairing DNA damage prior to replication and to protecting active replication forks in the presence of replisome-stalling lesions. Failure in these processes or in their coordination can lead to cancer and aging. The goal of this research is to use a combination of structural and functional approaches to investigate the protein-protein and protein- DNA interfaces required for coordinated damage recognition and repair in association with replication. We propose four Aims to examine keystone proteins involved in excision repair pathways that remove damage prior to or in coordination with the replisome, and with proteins involved in fork stability. Aim 1 will investigate the structural and functional basis for roles of XPG in NER, BER, and replication-associated repair by atomic resolution studies of the structured endonuclease domain and by structural characterization of the relatively unstructured R- and C-terminus domains via their interaction with partner proteins RPA, ubiquitinated PCNA, and DNA Ligase I. Aim 2 will investigate the structural and functional basis for the role of the annealing helicase, SMARCAL1, with RPA at stalled replication forks. Since SMARCAL1 is the first annealing helicase demonstrated to act in maintaining genome integrity at stalled forks, it is critically important to understand its function at a mechanistic level. Aim 3 will investigate the structural biology of NEIL1-initiated BER of oxidized bases, through interactions with FEN-1 and XPG, XRCC1/Ligase III, and with RPA. Aim 4 will focus on PNKP, which has kinase and phosphatase activities critical for both single-strand and double-strand break repair processes and which is also an essential component of NEIL-directed BER. We will investigate PNKP phosphatase domain substrate binding, structurally characterize PNKP interaction with a specific inhibitor of its phosphatase activity, and interrogate the interaction of PNKP with XRCC1/Ligase III. The proposed studies are built upon major findings and collaborations generated during the previous funding period in SBDR2. They include experimental interactions and substantial synergy with Projects 1, 3, 4, and 6, as well as with the EMB and SCB Cores. The anticipated outcome of these studies is a much more detailed molecular picture of the protein-protein and protein-DNA complexes involved in replication-associated repair. The information generated will elucidate the mode of action of a promising DNA repair inhibitor and contribute to its optimization, and will define new potential targets for novel cancer therapies.